Compositions and methods for treatment of glioma
In certain embodiments the present invention provides methods useful in the treatment of glioma, such as glioblastoma, such methods comprising administering to a subject in need thereof a CSF-IR inhibitor together with one or more additional active agents such as IGF-IR inhibitors, PI3K inhibitors, IL4 inhibitors, NFAT inhibitors, and/or Stat6 inhibitors. In certain embodiments the present invention provides pharmaceutical compositions comprising a CSF-IR inhibitor together with one or more of such additional active agents.
This application is a U.S. National Stage entry under 35 U.S.C. § 371 of International Patent Application No. PCT/US2016/031431, filed May 9, 2016, which claims the benefit of priority of U.S. Provisional Patent Application No. 62/158,671, filed on May 8, 2015, the content of which is hereby incorporated by reference in its entirety.
STATEMENT OF GOVERNMENT SUPPORTThis invention was made with government support under grant number CA181355 awarded by the National Institutes of Health. The government has certain rights in the invention.
INCORPORATION BY REFERENCEFor countries that permit incorporation by reference, all of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention.
BACKGROUNDGlioblastoma multiforme (GBM) is the most common and highly aggressive primary brain tumor, representing approximately 50% of all brain tumors. With standard of care therapy, which currently includes surgery, radiation, and temozolomide chemotherapy, patient prognosis is poor with an average survival of 12-15 months. Despite a clear and urgent need to improve treatment options for patients, most therapies aimed at directly targeting glioma cells in GBM have failed, largely due to substantial genetic and tumor cell heterogeneity, and a high propensity for recurrence.
As an alternative to tumor-targeted therapy, targeting the tumor microenvironment (TME) has been shown to be an effective therapeutic strategy in several tumor types, including brain tumors. Brain-resident and bone marrow-derived tumor-associated macrophages (TAMs) are the predominant immune cell population in GBMs comprising up to 30% of the bulk tumor mass. In many types of cancer, including glioma, high TAM numbers are associated with high grade tumors and poor patient prognosis.
Macrophages depend on colony stimulating factor (CSF)-1 for their differentiation and survival, and thus strategies to target macrophages often include blockade of the CSF-1 receptor (CSF-1R). It was previously shown that blocking CSF-1R with the compound BLZ945 in advanced, high-grade, gliomas resulted in rapid tumor de-bulking by more than 30% after just 7 days of treatment (Pyonteck et al., “CSF-1R inhibition alters macrophage polarization and blocks glioma progression;” Nat. Med. 2013 October; 19(10): pp. 1264-72). However, prior to the present invention the efficacy of CSF-1R inhibitors such as BLZ945 in longer-term treatment of high-grade glioma was unknown. Similarly, prior to the present invention it was not known whether longer-term treatment of GBM with CSF-1R inhibitors, such as BLZ945, might result in the emergence of drug resistance, and if so, what strategies might be available to ameliorate such drug resistance.
SUMMARY OF THE INVENTIONThe present invention is based, in part, on a series of important discoveries relating to the use of CSF-1R inhibitors for the treatment of glioma. As described in more detail in the Examples section of this patent specification, it has now been discovered that, despite good short-term therapeutic effects, in approximately half of subjects the efficacy of CSF-1R inhibitor treatment is significantly reduced during longer-term treatment regimens, with significant tumor recurrence and the development of drug resistance. The studies presented herein found that, while during the initial stages of CSF-1R treatment gliomas regressed (i.e. there was a decrease in tumor volume), the gliomas then entered a period of dormancy (with no significant change in tumor volume), which was then followed by a period of recurrence or rebound during which gliomas significantly increased in size—despite continued CSF-1R inhibitor treatment. It was found that this acquired CSF-1R inhibitor resistance was mediated by elevated phosphatidylinositol 3-kinase (“PI3-kinase or “PI3K”) signaling in the rebounding tumors, driven by a paracrine interaction between elevated insulin-like growth factor 1 (“IGF-1”) produced by tumor-associated macrophages (“TAMs”), and elevated IGF-1 receptors (“IGF-1R”) on the tumor cells. In particular, it was found that IGF-1 is upregulated in TAMs in response to IL4 secretion, in part via activation of NFAT and Stat6 signaling, and that the secreted IGF-1 results in activation of IGF-1R and PI3K signaling in glioma cells, supporting tumor growth and malignancy. Building on these discoveries, it was found that therapy with various different active agents acting at various steps in this signaling process (including IGF-1R inhibitors, PI3K inhibitors, NFAT inhibitors, and Stat6 inhibitors, or various combinations thereof) resulted in improved CSF-1R inhibitor sensitivity and significantly increased long-term survival. It was also found that treatment of naïve gliomas (i.e. gliomas that had not been treated with CSF-1R inhibitors) with some of these agents also resulted in a significant survival benefit—however, these effects were not as substantial as the survival benefits observed in rebound tumors following CSF-1R inhibition.
Accordingly, in some embodiments the present invention provides various methods useful for the treatment of glioma.
In some such embodiments such methods comprise administering to a subject in need thereof an effective amount of: a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and one or more agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor.
In other such embodiments such methods comprise administering to a subject an effective amount of: a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and two or more agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor. In some such embodiments, the subject is treated by administering an effective amount of a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), and a PI3K signaling pathway inhibitor (such as a PI3K inhibitor). In other such embodiments, the subject is treated by administering an effective amount of a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and both an NFAT inhibitor and a Stat6 inhibitor.
In other embodiments the present invention provides methods for the treatment of glioma, such methods comprising administering to a subject in need thereof an effective amount of one or more agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor.
Similarly, in some embodiments the present invention provides methods for the treatment of glioma, such methods comprising administering to a subject in need thereof an effective amount of two or more agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor. In some such embodiments the present invention provides methods for the treatment of glioma comprising administering to a subject in need thereof an effective amount of both an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), and a PI3K signaling pathway inhibitor (such as a PI3K inhibitor). Similarly, in other such embodiments the present invention provides methods for the treatment of glioma comprising administering to a subject in need thereof an effective amount of both an NFAT inhibitor and a Stat6 inhibitor.
In some embodiments the present invention provides pharmaceutical compositions, such as those that may be useful in the treatment of glioma.
In some such embodiments the pharmaceutical compositions comprise both a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and one or more additional agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor, a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor.
In other such embodiments the pharmaceutical compositions comprise both a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and two or more additional agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor, a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor. For example, in some such embodiments such compositions comprise a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), and a PI3K signaling pathway inhibitor (such as a PI3K inhibitor). In other such embodiments, the compositions comprise a CSF-1R signaling pathway inhibitor (such as a CSF-1R inhibitor), and both an NFAT inhibitor and a Stat6 inhibitor.
In other such embodiments the pharmaceutical compositions comprise two or more agents selected from the group consisting of: an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), a PI3K signaling pathway inhibitor (such as a PI3K inhibitor), an IL4 inhibitor, an NFAT inhibitor, and a Stat6 inhibitor. In some such embodiments the pharmaceutical compositions comprise both an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), and a PI3K signaling pathway inhibitor (such as a PI3K inhibitor). Similarly, in other such embodiments the pharmaceutical compositions comprise both an NFAT inhibitor and a Stat6 inhibitor.
Suitable inhibitors (such as CSF-1R inhibitors, IGF-1R inhibitors, PI3K inhibitors, IL4 inhibitors, NFAT inhibitors, and Stat6 inhibitors) that can be used in accordance with the methods and compositions of the present invention are known in the art and are described in the Detailed Description and Examples sections of this patent application. In some embodiments the inhibitors used are able to cross the blood-brain barrier, such that they may be administered to a subject systemically (for example via oral administration or intravenous administration). However, in some embodiments the inhibitors may not be able to cross the blood-brain barrier, or may have limited ability to cross the blood-brain barrier. In such embodiments, intracranial (e.g. intracerebral) administration may be used.
The present invention contemplates several different treatment regimens for administration of the various different inhibitors/active agents described herein. For example, in some embodiments administration of an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), PI3K signaling pathway inhibitor (such as a PI3K inhibitor), IL4 inhibitor, NFAT inhibitor, or Stat6 inhibitor is commenced approximately concurrently with administration of the CSF-1R inhibitor—without waiting for the development of dormancy or subsequent CSF-1R resistance.
However, in other embodiments the administration of an IGF-1/IGF-1R signaling pathway inhibitor (such as an IGF-1R inhibitor), PI3K signaling pathway inhibitor (such as a PI3K inhibitor), IL4 inhibitor, NFAT inhibitor, or Stat6 inhibitor is commenced subsequent to the commencement of CSF-1R inhibitor treatment, for example when the glioma is no longer regressing, or is dormant, or is rebounding/recurring, and/or has developed CSF-1R resistance.
In those embodiments that involve administration of IGF-1/IGF-1R signaling pathway inhibitors (such as IGF-1R inhibitors), PI3K signaling pathway inhibitors (such as PI3K inhibitors), IL4 inhibitors, NFAT inhibitors, or Stat6 inhibitors without CSF-1R inhibitor treatment, such inhibitors may be administered alone, or in conjunction with other non-CSF-1R-inhibitor agents, and treatment may be commenced on naïve tumors (e.g. tumors that have not yet been treated with another agent), or may be commenced after the tumor has been treated with other agents, for example after treatment with another agent has been commenced but the glioma is no longer regressing, or is dormant, or is rebounding/recurring, and/or has developed resistance to the other agent.
In some embodiments the methods of the present invention may be used to treat subjects that have previously been treated with a CSF-1R inhibitor or other agent useful for the treatment of glioma, for example subjects having a glioma that was previously sensitive to CSF-1R inhibitor treatment (or treatment with another agent), but that is no longer responsive to such treatment. Similarly, in some embodiments the methods of the present invention may be used to treat subjects having a glioma that is not, or does not appear to be, sensitive to CSF-1R inhibitor treatment or treatment with another agent.
The methods and compositions provided herein are directed to the treatment of glioma. In some embodiments the glioma may be a glioblastoma, such as a proneural glioblastoma or Glioblastoma Multiforme (GBM). In some embodiments the glioblastoma may be an astrocytoma or an oligodendroglioma.
A variety of subjects may be treated with the methods and compositions of the present invention. In some embodiments the subject is a mammal, for example a rodent (e.g. a mouse, rat, or guinea pig), a non-human primate, or a human. In addition to the specific treatment methods of the invention, subjects may also be treated using other treatment regimens known to be useful in the treatment of glioma, including, but not limited to, surgical methods (e.g. tumor resection surgery), radiation therapy, chemotherapy (for example using temozolomide), or anti-angiogenic therapy (for example using bevacizumab).
These and other embodiments are described in more detail elsewhere in this patent application, including in the Figures, Brief Description of the Figures, Detailed Description, and Examples sections of this patent application. Furthermore, it should be understood that variations and combinations of each of the embodiments described throughout this patent application are contemplated and are intended to fall within the scope of the present invention.
Some of the main embodiments of the present invention are described in the above Summary of the Invention section of this patent application, as well as in the Figures, Brief Description of the Figures, Examples, and Claims sections of this application. This Detailed Description section provides certain additional description relating to the compositions and methods of the present invention, and is intended to be read in conjunction with all other sections of the present patent application. The sub-headings provided below, and throughout this patent disclosure, are not intended to denote limitations of the various aspects or embodiments of the invention, which are to be understood by reference to the specification as a whole.
Definitions & Abbreviations
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably
Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form unless otherwise stated. Numeric ranges provided herein are inclusive of the numbers defining the range. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number.
The acronym “CSF-1” refers to colony stimulating factor 1. The acronym “CSF-1R” refers to the CSF-1 receptor.
The acronym “PI3K” refers to phosphatidylinositol 3-kinase.
The acronym “IGF-1” refers to insulin-like growth factor 1. The acronym “IGF-1R” refers to the IGF-1 receptor.
The acronym “IL4” refers to interleukin 4.
The acronym “NFAT” refers to nuclear factor of activated T-cells—a family of transcription factors.
The acronym “Stat6” refers to signal transducer and activator of transcription 6—a member of the Stat family of transcription factors.
An “active agent” is an agent (e.g. a small molecule, or a protein/peptide (such as an antibody)), for example as described and/or claimed herein, that has the recited activity—such as CSF-1R signaling pathway inhibitory activity, IGF-1/IGF-1R signaling pathway inhibitory activity, PI3K signaling pathway inhibitory activity, IL4 inhibitory activity, NFAT inhibitory activity, or Stat6 inhibitory activity. “Active agents” include, but are not limited to, the specific inhibitors described in this patent disclosure. It is also contemplated that, in each of the embodiments of the present patent disclosure that involve use of specified active agents, analogues, variants, or derivatives of each of such specified active agents can be used. One of skill in the art can readily determine whether an analogue, variant, or derivative of any of such specified active agent is suitable for use in accordance with the compositions and methods of the present invention, for example based on whether the analogue, variant, or derivative has one or more of the desired activities, such as, for example, CSF-1R inhibitory activity, IGF-1R inhibitory activity, PI3K inhibitory activity, IL4 inhibitory activity, NFAT inhibitory activity, and/or Stat6 inhibitory activity.
As mentioned above, in some embodiments active agents can be antibodies. The term “antibody,” as used herein, encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv, and single chain Fv (scFv) fragments, single-domain antibodies (sdAb or nanobodies)), fusion proteins comprising an antigen determination portion of an antibody, bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule(s) comprising an antigen recognition site—so long as the antibodies have the desired and/or recited biological activity—such as CSF-1R inhibitory activity, IGF-1R inhibitory activity, PI3K inhibitory activity, L4 inhibitory activity, NFAT inhibitory activity, and/or Stat6 inhibitory activity. Various different types of antibody fragments, and methods of making and using such antibody fragments, are known in the art. See, for example, Fridy et al., Nature Methods. 2014 December; 11(12):1253-60 (the contents of which are hereby incorporated by reference) for a description of the production of nanobody repertoires multi-specific antibodies. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked, or conjugated to other molecules such as toxins, radioisotopes, or any of the other specific molecules recited herein.
The terms “inhibit,” “block,” “reduce,” and “suppress” are used interchangeably and refer to any statistically significant decrease in biological activity, including—but not limited to—full blocking of the activity.
Various other terms are defined elsewhere in this patent disclosure, where used. Furthermore, terms that are not specifically defined herein may be more fully understood in the context in which the terms are used and/or by reference to the specification in its entirety. Where no explicit definition is provided, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains.
CSF-1R Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more CSF-1R signaling pathway inhibitors (such CSF-1R inhibitors). In certain other embodiments the present invention provides compositions comprising one or more CSF-1R signaling pathway inhibitors (such CSF-1R inhibitors). In some of such embodiments, any suitable CSF-1R signaling pathway inhibitor can be used. In some embodiments the suitability of a CSF-1R signaling pathway inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature (for example from published studies demonstrating anti-tumor activity, such as anti-glioma activity), or may be ascertained by employing various assays for anti-tumor activity (such as anti-glioma activity), such as those described in the Examples section of the present patent application. Several CSF-1R inhibitors that are known in the art can be used in conjunction with the present invention. For example, in some embodiments, any one or more of the following CSF-1R inhibitors (or classes of inhibitors) may be used: BLZ945, GW2580, ABT-869 (Linifanib), OSI-930, CEP-32496, AC708, PLX3397, AZD6495, CYC10268, IMC-CS4, RG7115, pyridyl bisamides, thiazolyl bisamides, 6-O-substituted benzoxazoles, and 6-O-substituted benzothiazoles. In some embodiments, any suitable variant, analogue or derivative of any one of such CSF-1R inhibitors may be used. In some embodiments the CSF-1R inhibitor may be a small molecule, or an antibody, or any other suitable agent that has CSF-1R inhibitory activity. In some embodiments the CSF-1R inhibitor used is one that can permeate the blood-brain barrier. In some embodiments the CSF-1R inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the CSF-1R inhibitor the ability to permeate the blood-brain barrier. In some embodiments, the CSF-1R inhibitor is one of those described in WO 2012/151541, WO 2007/121484, US 2015/0080556, US 2014/0336363, US 2014/0065141, US 2014/0057972, US 2013/0005949, US 2012/0225861, US 2010/0280006, US 2010/0261679, US 2010/0130490, US 2009/0054411, US 2008/0045528, or US 20040014774. In some embodiments, the CSF-1R inhibitor used is BLZ945, or an analogue, variant, or derivative thereof. BLZ945 (or 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino) benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide, CAS Registry No.: 953769-46-5) is an orally active, potent and selective CSF-1R inhibitor, which inhibits CSF-1R activity with an IC50 of 1 nM and is more than 1000-fold selective against its closest receptor tyrosine kinase homologs c-KIT and Platelet-derived Growth Factor Receptor beta (PDGFRb). See Pyonteck et al., “CSF-1R inhibition alters macrophage polarization and blocks glioma progression;” Nat. Med. 2013 October; 19(10):pp. 1264-72, the contents of which are hereby incorporated by reference.
IGF-1R Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more IGF-1/IGF-1R signaling pathway inhibitors (such as IGF-1R inhibitors). In certain other embodiments the present invention provides compositions comprising one or more IGF-1/IGF-1R signaling pathway inhibitors (such as IGF-1R inhibitors). In some of such embodiments, any suitable IGF-1/IGF-1R signaling pathway inhibitor can be used. In some embodiments the suitability of an IGF-1R inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature (for example from published studies demonstrating anti-IGF-1R activity), or may be ascertained by employing various assays for IGF-1R activity known in the art, or may be ascertained by employing one of the assays described in the Examples section of the present patent application to demonstrate improved anti-tumor activity when the IGF-1R inhibitor is employed in conjunction with a CSF-1R inhibitor. Several IGF-1R inhibitors that are known in the art can be used in conjunction with the present invention. For example, in some embodiments, any one or more of the following IGF-1R inhibitors (or classes of inhibitors) may be used: OSI906 (linsitinib), NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, picropodophyllin (PPP), GSK1838705A, AG-1024, PQ401, and BMS-754807. In some embodiments, any suitable variant, analogue or derivative of any one of such IGF-1R inhibitors may be used. In some embodiments the IGF-1R inhibitor may be a small molecule, or an antibody, or any other suitable agent that has IGF-1R inhibitory activity. In some embodiments the IGF-1R inhibitor used is one that can permeate the blood brain barrier. In some embodiments the IGF-1R inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the IGF-1R inhibitor the ability to permeate the blood-brain barrier. In some embodiments, the IGF-1R inhibitor is one of those described in US 2009/0054508, US 2010/0226884 or US 2014/0086830. In some embodiments, the CSF-1R inhibitor used is OSI906 (CAS Registry No. 867160-71-2), or an analogue, variant, or derivative thereof. See Mulvihill et al. “Discovery of OSI-906: A Selective and Orally Efficacious Dual Inhibitor of the IGF-1 Receptor and Insulin Receptor;” Future Med. Chem. 2009 September; 1(6): pp. 1153-71, the contents of which are hereby incorporated by reference.
PI3K Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more PI3K signaling pathway inhibitors (such as PI3K inhibitors). In certain other embodiments the present invention provides compositions comprising one or more PI3K signaling pathway inhibitors (such as PI3K inhibitors). In some of such embodiments, any suitable PI3K signaling pathway inhibitor can be used. In some embodiments the suitability of a PI3K inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature (for example from published studies demonstrating anti-PI3K activity), or may be ascertained by employing various assays for PI3K activity known in the art, or may be ascertained by employing one of the assays described in the Examples section of the present patent application to demonstrate improved anti-tumor activity when the PI3K inhibitor is employed in conjunction with a CSF-1R inhibitor. Several PI3K inhibitors that are known in the art can be used in conjunction with the present invention. For example, in some embodiments, any one or more of the following PI3K inhibitors (or classes of inhibitors) may be used: BKM120 (NVP-BKM120, buparlisib), idelaisib, SAR245409, SAR245408, BYL-719, GDC-0980, GDC-0941, wortmannin, Ly294002, demethoxyviridin, perifosine, delalisib, PX-866, IPI-145, BAY 80-6946, BEZ235, RP6530, TGR1202, RP5264, SF1126, INK1117, palomid529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477, CUDC-907, and AEZS-136. In some embodiments, any suitable variant, analogue or derivative of any one of such PI3K inhibitors may be used. In some embodiments the PI3K inhibitor may be a small molecule, or an antibody, or any other suitable agent that has PI3K inhibitory activity. In some embodiments the PI3K inhibitor used is one that can permeate the blood brain barrier. In some embodiments the PI3K inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the PI3K inhibitor the ability to permeate the blood-brain barrier. In some embodiments, the PI3K inhibitor used is BKM120 (CAS Registry No. 944396-07-0), or an analogue, variant, or derivative thereof.
IL4 Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more IL4 signaling pathway inhibitors (such IL4 inhibitors). In certain other embodiments the present invention provides compositions comprising one or more IL4 signaling pathway inhibitors (such IL4 inhibitors). In some of such embodiments, any suitable IL4 signaling pathway inhibitor can be used. In some embodiments the suitability of an IL4 signaling pathway inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature, or may be ascertained by employing various assays for anti-tumor activity (such as anti-glioma activity), such as those described in the Examples section of the present patent application. Several IL4 inhibitors that are known in the art can be used in conjunction with the present invention. In some embodiments, any suitable variant, analogue or derivative of any one of such IL4 inhibitors may be used. In some embodiments the IL4 inhibitor may be a small molecule, or an antibody, or any other suitable agent that has IL4 inhibitory activity. In some embodiments the IL4 inhibitor used is one that can permeate the blood-brain barrier. In some embodiments the IL4 inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the IL4 inhibitor the ability to permeate the blood-brain barrier.
NFAT Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more NFAT pathway inhibitors (such NFAT inhibitors). In certain other embodiments the present invention provides compositions comprising one or more NFAT pathway inhibitors (such NFAT inhibitors). In some of such embodiments, any suitable NFAT pathway inhibitor can be used. In some embodiments the suitability of an NFAT signaling pathway inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature, or may be ascertained by employing various assays for anti-tumor activity (such as anti-glioma activity), such as those described in the Examples section of the present patent application. Several NFAT inhibitors that are known in the art can be used in conjunction with the present invention. For example, in some embodiments, any one or more of the following NFAT inhibitors (or classes of inhibitors) may be used: Met-Ala-Gly-Pro-His-Pro-Val-Ile-Val-Ile-Thr-Gly-Pro-His-Glu-Glu (i.e. VIVIT peptide, CAS registry number 249537-73-3), 11R-VIVIT peptide, FK506, and INCA-6. In some embodiments, any suitable variant, analogue or derivative of any one of such NFAT inhibitors may be used. In some embodiments the NFAT inhibitor may be a small molecule, or an antibody, or any other suitable agent that has NFAT inhibitory activity. In some embodiments the NFAT inhibitor used is one that can permeate the blood-brain barrier. In some embodiments the NFAT inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the NFAT inhibitor the ability to permeate the blood-brain barrier.
Stat6 Inhibitors
In certain embodiments the present invention provides methods for the treatment of gliomas that comprise administration of one or more Stat6 pathway inhibitors (such Stat6 inhibitors). In certain other embodiments the present invention provides compositions comprising one or more Stat6 pathway inhibitors (such Stat6 inhibitors). In some of such embodiments, any suitable Stat6 pathway inhibitor can be used. In some embodiments the suitability of an Stat6 signaling pathway inhibitor for use in accordance with the methods of the present invention may be ascertained from the literature, or may be ascertained by employing various assays for anti-tumor activity (such as anti-glioma activity), such as those described in the Examples section of the present patent application. Several Stat6 inhibitors that are known in the art can be used in conjunction with the present invention. For example, in some embodiments, the Stat6 inhibitor AS1517499 may be used. In some embodiments, any suitable variant, analogue or derivative of any one of such Stat6 inhibitors may be used. In some embodiments the Stat6 inhibitor may be a small molecule, or an antibody, or any other suitable agent that has Stat6 inhibitory activity. In some embodiments the Stat6 inhibitor used is one that can permeate the blood-brain barrier. In some embodiments the Stat6 inhibitor may be linked to, or capable or co-delivery with, another agent that can confer upon the Stat6 inhibitor the ability to permeate the blood-brain barrier.
Methods of Treatment & Prevention
In certain embodiments the present invention provides methods for the treatment and/or prevention of gliomas in subjects in need thereof, such methods comprising administering to the subject an effective amount of certain active agents.
The term “glioma” is used herein in accordance with its normal usage in the art and includes a variety of different tumor types, including, but not limited to gliomas, glioblastoma multiforme (GBM), astrocytomas, and oligodendrogliomas.
As used herein, the terms “treat,” “treating,” and “treatment” encompass a variety of activities aimed at achieving a detectable improvement in one or more clinical indicators or symptoms associated with glioma. For example, such terms include, but are not limited to, reducing the rate of growth of a glioma (or of glioma cells, or of other cells within a glioma), halting the growth of a glioma (or of glioma cells, or of other cells within a glioma), causing regression of a glioma (or of glioma cells, or of other cells within a glioma), reducing the size of a glioma (for example as measured in terms of tumor volume or tumor mass), reducing the grade of a glioma, eliminating a glioma (or glioma cells, or other cells within a glioma), preventing, delaying, or slowing recurrence (rebound) of a glioma, improving symptoms associated with glioma, improving survival from glioma, inhibiting or reducing spreading of a glioma (e.g. metastases), and the like.
For each of the methods and compositions described herein that are directed to “treatment” of glioma, in some embodiments such methods and compositions can be employed for “prevention” of glioma. As used herein the terms “prevent” and/or “prevention” refer to prophylactic or preventative measures that prevent and/or slow the development of a glioma. Thus, those in need of prevention include those at risk of, or susceptible to, glioma. In certain embodiments, a glioma is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the glioma, or a later onset of symptoms associated with the glioma, than a patient who has not been subject to the methods and/or compositions of the invention.
In certain embodiments the methods of treatment and/or prevention provided herein may be employed together with other glioma treatment and/or prevention methods, including, but not limited to, surgical methods (e.g. for tumor resection), radiation therapy methods, treatment with chemotherapeutic agents (e.g. temozolomide, carmustine (BCNU), or cisplatin), treatment with antiangiogenic agents (e.g. bevacizumab), or treatment with tyrosine kinase inhibitors (such as gefitinib or erlotinib). Similarly, in certain embodiments the methods of treatment and/or prevention provided herein may be employed together with procedures used to monitor disease status/progression, such as biopsy methods and diagnostic methods (e.g. MM methods or other imaging methods).
Subjects
The terms “subject,” “individual,” and “patient”—which are used interchangeably herein, are intended to refer to any subject, preferably a mammalian subject, and more preferably still a human subject, for whom therapy or prophylaxis desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, mice, rats, guinea pigs, and the like. In most of the embodiments of the present invention the subject has, or is suspected of having, a glioma—such as glioblastoma multiforme (GBM), an astrocytoma, or an oligodendroglioma.
Administration Routes
In carrying out the treatment and/or prevention methods described herein, any suitable method or route of administration can be used to deliver the active agents.
In some embodiments systemic administration may be employed. “Systemic administration” means that the active agent is administered such that it enters the circulatory system, for example, via enteral, parenteral, inhalational, or transdermal routes. Enteral routes of administration involve the gastrointestinal tract and include, without limitation, oral, sublingual, buccal, and rectal delivery. Parenteral routes of administration involve routes other than the gastrointestinal tract and include, without limitation, intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous.
In some embodiments (including, but not limited to, those in which one or more of the agents used is not able to permeate the blood-brain barrier), local administration may be employed.
“Local administration” means that a pharmaceutical composition is administered directly to where its action is desired (e.g., at or near the site of a glioma), for example via intracranial (e.g. intracerebral) delivery, such as via direct intratumoral injection. For example, ins some embodiments pressure-driven infusion through an intracranial catheter, also known as convection-enhanced delivery (CED) may be used.
It is within the skill of one of ordinary skill in the art to select an appropriate route of administration taking into account the nature of the specific active agent being used and nature of the specific glioma to be treated.
Effective Amounts
As used herein the terms “effective amount” or “therapeutically effective amount” refer to an amount of an active agent as described herein that is sufficient to achieve, or contribute towards achieving, one or more desirable clinical outcomes, such as those described in the “treatment” and “prevention” descriptions above. An appropriate “effective” amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the desired route of administration (e.g. systemic vs. intracranial), desired frequency of dosing, etc. Furthermore, an “effective amount” may be determined in the context of the co-administration method to be used. For example, rather than perform dosing studies using an IGF-1R inhibitor alone, or a PI3K inhibitor alone, dosing studies may be performed in conjunction with administration of a CSF-1R inhibitor, because, as described herein, the effects of such agents may be synergistic. One of skill in the art can readily perform such dosing studies to determine appropriate doses to use, for example using assays such as those described in the Examples section of this patent application—which involve administration of a CSF-1R inhibitor together with either an IGF-1R inhibitor or a PI3K inhibitor.
Pharmaceutical Compositions
In certain embodiments, the present invention provides compositions, for example pharmaceutical compositions. The term “pharmaceutical composition,” as used herein, refers to a composition comprising at least one active agent as described herein, and one or more other components useful in formulating a composition for delivery to a subject, such as diluents, buffers, saline (such as phosphate buffered saline), cell culture media, carriers, stabilizers, dispersing agents, suspending agents, thickening agents, excipients, preservatives, and the like. “Pharmaceutical compositions” permit the biological activity of the active agent, and do not contain components that are unacceptably toxic to the living subject to which the composition would be administered.
Pharmaceutical compositions can be in numerous dosage forms, for example, tablet, capsule, liquid, solution, soft-gel, suspension, emulsion, syrup, elixir, tincture, film, powder, hydrogel, ointment, paste, cream, lotion, gel, mousse, foam, lacquer, spray, aerosol, inhaler, nebulizer, ophthalmic drops, patch, suppository, and/or enema. The choice of dosage forms and excipients will depends upon the active agent to be delivered and the specific disease or disorder to be treated or prevented, and can be selected by one of ordinary skill in the art without having to engage in any undue experimentation.
The present invention can be further understood a by reference to the following “Example”—which is non-limiting and provided for illustration purposes only. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
EXAMPLENumbers in parentheses following text in this Example represent the numbered references provided in the Reference List that follows.
Therapies targeted against the tumor microenvironment (TME) represent a promising approach for treating cancer. This appeal arises in part from the decreased likelihood of acquired resistance through mutations in target TME cells, as is frequently observed with cancer cell-targeted therapies. As multiple TME-directed therapies are currently advancing through different clinical trials (1, 2), this necessitates an understanding of potential mechanisms of intrinsic or acquired resistance. This experiments presented herein address this issue by investigating whether resistance to a macrophage-targeted therapy emerges during the course of long-term trials in various preclinical models of high-grade glioma (glioblastoma multiforme; GBM).
GBM is the most common and aggressive adult primary brain tumor, and survival is only minimally prolonged by current standard of care treatment, including surgery, radiation and temozolomide chemotherapy (3). Accordingly, targeting the glioma TME is emerging as a promising alternative therapeutic strategy. In GBM, tumor-associated macrophages and microglia (TAMs) comprise up to 30% of the bulk tumor mass (4). In many cancers, including glioma, elevated TAM numbers are associated with high grade and poor patient prognosis (4-7). As such, targeting TAMs in GBM represents an attractive therapeutic approach.
Macrophages critically depend on colony stimulating factor-1 (CSF-1) for multiple functions; consequently, strategies to target TAMs often include CSF-1 receptor (CSF-1R) blockade (8-10). In clinical trials, several approaches to inhibit CSF-1R are currently being employed including antibodies and small molecules (7, 11, 12). However, the long-term effects of these agents on clinical outcome are still under evaluation, and thus gaining insight into potential mechanisms of drug resistance and/or inefficacy is now critical.
In the work described herein a potent and highly selective small-molecule CSF-1R inhibitor, BLZ945, as used. It has been shown that BLZ945 blocks early gliomagenesis, while short-term treatment of advanced, high-grade glioma causes robust tumor debulking after just 7 days (8). Interestingly, CSF-1R inhibition has no direct effect on glioma cell viability, as these cells do not express CSF-1R in the models used here. Instead, glioma TAMs remain abundant and become anti-tumorigenic in response to treatment, by downregulating markers of M2-like macrophage polarization/alternative activation and adopting a pronounced phagocytic phenotype (8). This example addresses the unanswered question of whether long-term CSF-1R inhibition in aggressive late-stage GBM has a sustainable anti-tumorigenic effect, or instead leads to acquired resistance.
A Subset of GBMs Develop Resistance to CSF-1R Inhibition in Long-Term Preclinical Trials
The kinetics of GBM response to continuous long-term BLZ945 treatment was analyzed using a transgenic platelet-derived growth factor-driven glioma (PDG) model (RCAS-hPDGF-B/Nestin-Tv-a; Mk4a/Arf−/−) (8, 13) (
Following this regression phase, all BLZ945-treated tumors entered a dormancy phase, lasting for ≥4 weeks (
Experiments were performed to determine how this resistance to the CSF-1R inhibitor emerged, and several time points were chosen throughout the long-term trial for comparison, including Veh (vehicle; 20% Captisol until symptomatic), 7d (BLZ945-responsive, regressing), 28d (BLZ945-responsive, dormant), Reb (BLZ945-resistant, actively rebounding), and EP (26-week endpoint, stably regressed) (
Glioma Cells Resistant to CSF-1R Inhibition In Vivo Exhibit Elevated PI3K Signaling.
To determine the mechanism by which tumor cells acquire resistance, array comparative genomic hybridization (aCGH) analyses were performed and no copy number alterations were found in primary rebound glioma tumorsphere lines (passage 1;
To investigate whether PI3K signaling is functionally important in driving recurrence, a preclinical intervention trial was performed. PDG mice bearing high-grade gliomas were treated with BLZ945 until they showed tumor rebound by MRI, at which point BKM120 treatment was performed (
To determine if recurrence could be prevented by earlier PI3K inhibition, during the initial dormancy phase, GBM-bearing PDG mice were treated with BLZ945 alone for 28d, at which point BKM120 was added until the trial endpoint (
The finding that PI3K activation underlies resistance to CSF-1R inhibition was intriguing in light of the high frequency of mutations in the PI3K pathway in glioma patients (16). Therefore studies were performed to investigate whether genetic mutations in PI3K/PTEN would similarly confer a resistance-like phenotype in mouse models, which could potentially be informative in the clinical setting. To address this question, BLZ945 efficacy was compared in two additional RCAS-hPDGF-B/Nestin-Tv-a GBM models harboring distinct clinically-relevant oncogenic alterations besides Ink4a/Arf loss, including Pten deletion (Pten KO model) or p53 knockdown (p53 KD model; see methods). After 2 weeks of treatment, it was found that BLZ945 efficacy in the p53 KD model (56% reduction of tumor volume) was comparable to that of the PDG model (62% reduction). However, CSF-1R inhibition was less potent in the Pten KO model over the same time period (3% reduction;
Resistance to CSF-1R Inhibition is Mediated by the Microenvironment
Next studies were performed to investigate how PI3K was activated in rebound tumors, and to explore whether resistance to BLZ945 was tumor cell-intrinsic or -extrinsic. It was previously established that BLZ945 does not directly affect glioma cell lines in culture (8), and it is demonstrated herein that CSF-1R inhibition also has no direct effect on viability of a panel of primary cell lines derived from rebound tumors (
The TME was analyzed in recurrent disease to determine how resistance to CSF-1R inhibition develops. It was found that rebounding tumors always emerged adjacent to regions of glial scarring, characterized by reactive astrocytes, calcium deposition, and relatively low vascularity associated with elevated hypoxia (
Rebound TAMs Adopt a Wound-Associated Signature that is Driven by Enhanced Interleukin-4 (IL-4) Signaling
During gliosis, activated macrophages play a central role in providing growth factors and signaling molecules to nearby astrocytes and neurons, to form a reactive barrier that limits the extent of tissue damage in the brain (18, 20). Given that BLZ945 is a macrophage-targeted drug, TAM numbers and phenotype were analyzed in rebound tumors. It has previously been shown that TAMs are not depleted in the glioma TME in a 1-week trial with BLZ945, but rather downregulate expression of M2-like genes and increase phagocytosis of tumor cells (8, 21). Consistently, it is shown herein that TAMs are still present in 7d, 28d, EP or Reb tumors (
To assess potential differences in activation states, TAMs were FACS-purified from Veh, EP and Reb tumors, and RNA-seq was performed. Principal component analysis confirmed distinct global gene expression profiles for Veh, EP and Reb TAMs (
Given the similarities in alternative activation between Veh and Reb TAMs according to the M1/M2-like paradigm (26), yet clear differences in drug response between treatment-naïve and rebound tumors, a more fine-tuned approach was employed to defining macrophage phenotype. A spectrum model of macrophage activation was computationally interrogated, defined by gene sets that are altered in response to different stimuli, including interferon γ (IFNγ), IL4, TNFα, transforming growth factor β1 (TGFβ1), IL1β, and two toll-like receptor (TLR) agonists specific for TLR2 (macrophage-activating lipopeptide 2; MALP2) and TLR9 (unmethylated CpG-containing oligonucleotide; CPG) (27, 28). It was determined that IL4- and TGFβ1-targeted gene sets were significantly enriched in Reb TAMs compared to Veh TAMs (
To determine the cellular source of IL4 in this model, multicolor flow cytometry was used to immune-profile Veh, EP, and Reb tumors using a panel of myeloid (CD11b, Gr1, Ly6G, Ly6C, CD11c, Tie2, MHCII) and lymphoid (CD19, CD3, CD4, CD8, FoxP3) cell markers. While it was found that few cell types were uniquely enriched in rebound tumors (
The IGF-1/IGF-1R Signaling Axis is Induced in Rebound Gliomas
Next studies were performed to investigate how IL4 and wound-associated gene expression might be connected to PI3K signaling in rebound tumors. Differential gene expression analysis revealed that TAM-derived Igf1 was one of the most significantly upregulated genes in Reb TAMs compared to EP TAMs, which was confirmed in comparisons with Veh TAMs or 28d TAMs (
To assay the effects of IGF-1 specific to the rebound setting, multiple approaches were used: first, through western blot analysis, higher baseline levels of p-IGF-1R in early-passage rebound tumor cell lines compared to cell lines that we were able to propagate from dormant tumors in culture were confirmed (
Next, to model the effects of macrophage-derived IGF-1 on rebound tumor cells, an ex vivo culture system was designed using primary glioma microenvironment cultures (GMECs). GMECs contain multiple cell types from the glioma TME when harvested at early-passage, including macrophages, T cells, astrocytes, among others (
To assess the relevance of the IGF-1/IGF-1R axis in human malignancy, IGF1 expression was evaluated in publicly available human GBM gene expression datasets. It was found that IGF1 expression was significantly correlated with macrophage-specific genes (CSF1R, CD68 and AIF1) and with genes associated with an M2-like phenotype (CD163 and MRC1) in TCGA GBM samples (
To determine if macrophages are the predominant source of IGF1 in humans, qRT-PCR was used to show that macrophages express high levels of IGF1 compared to different immune cell types, astrocytes, endothelial cells, and glioma cells (
Finally, publicly available datasets were used to assess survival correlations in patients. Kaplan Meier curves were generated using a median cutoff for IGF1 expression levels, and no significant difference in overall survival between IGF1high and IGF1low patients was found (
NFAT and Stat6 Cooperate to Regulate Igf1 Expression in Rebound TAMs
Transcription factor (TF) activity analysis was next utilized to identify putative transcriptional networks regulating Igf1 expression in rebound tumors. Seven TF families showed enriched activity in Reb TAMs compared to EP TAMs (
To further characterize the role of IL4-induced NFAT and/or Stat6 signaling in Igf1 regulation, a series of in vitro and in vivo experiments were performed. First, it was confirmed that IL4 strongly induced expression of Igf1 and the three representative alternative activation/wound-associated genes (Retnla, Chil3 and Ccl17) in WT BMDMs, and that this capacity was reduced in BMDMs from either Stat6−/− or Il4ra flox; LysM-cre mice (
Combination of CSF-1R and IGF-1R Inhibition Improves Outcome
To formally test the hypothesis that the IGF-1/IGF-1R axis may underlie resistance to CSF-1R inhibition, IGF-1R was targeted in vivo using both pharmacological and genetic approaches. First, preclinical intervention trials were designed that were similar to those used for BKM120, except with an inhibitor of IGF-1R (OSI906/Linsitinib). PDG mice were treated with BLZ945 until they showed signs of rebound by MRI, at which point intervention with OSI906 was commenced (
To determine if tumor outgrowth could be prevented by earlier IGF-1R inhibition, PDG mice with high-grade GBMs were treated continuously with BLZ945 alone until 28d, at which point OSI906 was added until the trial endpoint (
Given that pharmacological inhibition of IGF-1R using OSI906 cannot confirm whether tumor cell-specific blockade is sufficient to reduce recurrent disease, a genetic approach was utilized to target this receptor in glioma cells. U251 glioma cell lines were genetically engineered to express a doxycycline-inducible shRNA against IGF1R, and orthotopic xenograft experiments were performed (see
Discussion
This Example demonstrates, and elucidates the mechanisms underlying, the development of acquired resistance to CSF-1R inhibition in mouse models of GBM. While initial therapeutic response to CSF-1R inhibition is robust, rapid, and completely penetrant, it has been shown herein that approximately half of the animals eventually develop resistance, with rapidly progressing rebound tumors. In light of recent results from ongoing patient studies with CSF-1R inhibitors in glioma (46) and other cancers, these findings suggest the need to prepare for the emergence of therapeutic resistance to CSF-1R inhibitors in GBM in the clinical setting, and determine if other brain malignancies besides GBM will respond similarly to CSF-1R inhibition. While classical mechanisms of tumor cell-intrinsic resistance to cytotoxic and targeted agents have been well-defined, including aberrant drug metabolism and transport, drug target mutation, and activation of alternative survival pathways (47), it still remains unclear whether resistance to TME-directed therapies will follow similar principles. Given that TME-targeted agents are increasingly being evaluated in the clinic (1, 2), it will be critical to mechanistically define how resistance evolves in response to these therapies in order to provide long-term disease management for patients.
In light of this problem, the present Example identifies a mechanism of drug resistance that can circumvent therapeutic response to a TME-targeted therapy, and promote disease progression in the absence of tumor cell-intrinsic alterations. Specifically, the work presented herein demonstrated a heterotypic paracrine signaling interaction that is initiated by the TME, and that drives resistance to CSF-1R inhibition. In rebound tumors, it was found that IGF-1 is upregulated in TAMs in response to IL4 (possibly supplied by T cells or alternative cell types), in part via NFAT activation. IGF-1 secretion into the extracellular environment results in activation of IGF-1R and PI3K signaling in glioma cells, supporting tumor growth and malignancy (see model in
Importantly, the findings presented herein underscore the importance of bidirectional feedback between cancer cells and their microenvironment, and support the notion that while stromal cells are less susceptible to genetic mutation than cancer cells, a tumor can nonetheless persist by exploiting its extracellular environment to acquire a resistant phenotype. Thus, an integrated analysis of cancer cells with their microenvironment can be helpful in understanding both their parallel evolution during tumor progression, and their capacity for adaptation in the context of therapeutic intervention and the development of resistance.
Materials and Methods
Study Design.
The overall objective of these studies was to understand how resistance to CSF-1R inhibitors develops in high-grade glioma using various in vivo and in vitro models. Within the animal studies, mice were randomly assigned to different therapy groups, which included treatment with BLZ945 (a CSF-1R inhibitor) in combination with inhibitors of putative resistance pathways (e.g. IGF-1R or PI3K), versus single-agent or vehicle controls. Survival and disease progression were monitored using a combination of Mill, histology, flow cytometry, and gene expression analyses throughout all trials. To power these studies, sample size was predefined as at least n=3 independent experiments, replicates or samples for in vitro and in vivo experiments, and up to a maximum of n=90 mice for survival analyses. Replicate values are indicated for each experiment in the figure legend. All analyses were calculated in a blinded manner through numerical coding of samples. For all long-term survival trials, 26 weeks was selected as a predefined endpoint because mice in the Ink4a/Arf background develop spontaneous lymphomas and sarcomas beginning at ˜30 weeks of age. For in vivo trials, mice were considered outliers if they developed (i) spontaneous lymphomas (given this disposition in the Ink4a/Arf−/− background), (ii) early symptoms of tumor burden >1 week prior to trial enrollment, or (iii) development of hydrocephalus prior to trial enrollment.
Biologicals and Pharmaceuticals (Active Agents).
BLZ945 (Novartis) (8) was used for both in vitro and in vivo studies. 6,700 nM of BLZ945 was used for all in vitro experiments in tumor cells, versus an equal percent DMSO as a vehicle control. This concentration represents 100× the IC50 for BLZ945 in macrophages (8). For in vivo experiments, BLZ945 was obtained pre-formulated at 12.5 mg/ml. BLZ945 was administered p.o. 1× daily at 200 mg/kg, and 20% Captisol was used as a vehicle control. BKM120 (Buparlisib; 1 μM unless indicated otherwise), AEW541, ADW742, BMS754807 and OSI906 (Linsitinib; 10 μM unless indicated otherwise) (all from Selleckchem) were used in vitro up to 100 μM for dose response assays versus an equal percent DMSO as a vehicle control. BKM120 and OSI906 (ChemieTek) were used in vivo. BKM120 was formulated by dissolving 52 mg into 500 ml NMP, boiling, and then adding 9.5 ml of PEG300. BKM120 was administered p.o. 1× daily at 20 mg/kg, and NMP:PEG300 (1:19) was used as a vehicle control. Animals were dosed at 20 mg/kg to avoid off-target effects, as BKM120 binds tubulin at concentrations above 50 mg/kg in subcutaneous tumor models, but not below 40 mg/kg (15). OSI906 was formulated daily at 4 mg/ml in 25 mM tartaric acid with shaking and sonication for ˜15 min. OSI906 was administered p.o. 1× daily at 40 mg/kg, and 25 mM tartaric acid was used as a vehicle control. It was decided to treat at 40 mg/kg as the maximum tolerated dose for OSI906, 75 mg/kg (48), was found to be toxic within 4 days in these studies. For all combination trials, BLZ945 was administered in the morning, and BKM120 or OSI906 was administered in the evening. The NFAT inhibitor, INCA-6 (Tocris) was used at a concentration of 40 μM for in vitro use (49). For in vivo inhibition of NFAT signaling, FK506 was used to inhibit the activating interaction between calcineurin and NFAT, at a dose of 10 mg/kg (administered i.p. every 3 days) (50). The Stat6 inhibitor, AS1517499 (Axon Medchem) was used at a concentration of 50 nM for in vitro use, and dosed at 10 mg/kg for in vivo use (administered i.p. 2× weekly) (51). The vehicle control for FK506 and AS1517499 in vivo was 10% EtOH and 1% Tween-80 in PBS. For in vitro PCR assays, recombinant mouse IL4 (R&D Systems) was used at a concentration of 10 ng/ml, recombinant mouse TGFβ1 (R&D Systems) was used at a concentration of 50 ng/ml, and the TGFβ1 type 1 receptor inhibitor, SB431542 (Tocris), was used at a concentration of 10 μM. For ex vivo glioma microenvironment culture (GMEC) assays, a neutralization antibody against IGF-1 (R&D Systems) was used at a concentration of 0.5 μg/ml. For culture of macrophages in vitro, recombinant mouse CSF-1 was used at a concentration of 10 ng/ml. For in vitro assays using bone marrow-derived macrophages (BMDMs), CSF-1 supplementation was excluded from all experimental conditions.
Animals.
Crl:NU(NCr)-Foxn1nu immunodeficient mice (Charles River Laboratories) were used for orthotopic transplantation studies. NOD/CB17-Prkdcscid immunodeficient mice (The Jackson Laboratory) were used for orthotopic implantation of human cells. Three different transgenic mouse models expressing the avian tv-a receptor under the control of the nestin (N) promoter in either mixed strain or BL6 backgrounds were used (Ntv-a; Ink4a/Arf−/−, Ntv-a, and Ntv-a; Ptenflox), all previously described (13, 52-57). Stat6−/−, Il4raflox; LysM-cre, and wild-type (WT) C57BL/6 (BL6) mice were used for bone marrow isolations. All animal studies were performed after obtaining the necessary approvals.
PDG Mouse Model.
The initiation of PDGF-driven gliomas (PDG) with RCAS-hPDGF-B-HA in adult mice has been previously described (8, 13, 58). Briefly, Ntv-a; Ink4a/Arf−/− mice were fully anesthetized with ketamine/xylazine prior to surgery. Pain management included a 50 μl subcutaneous injection of bupivacaine (0.25%) at the surgical site prior to surgery, and an intraperitoneal injection of buprenorphine immediately following surgery. Mice were intracranially injected with DF-1:RCAS-hPDGF-B-HA cells (200,000 cells/1 μl) between 5-6 weeks of age using a fixed stereotactic apparatus (Stoelting). Injections were made into the right frontal cortex, approximately 1.5 mm lateral and 1 mm caudal from bregma, and at a depth of 2 mm into the subventricular zone (SVZ). In this model, injection into the SVZ induces tumors with low latency (4-5 weeks), 100% penetrance, and histological features characteristic of patient GBM including microvascular proliferation and pseudopalisading necrosis (13). The incision was sealed using Vetbond tissue adhesive (3M). Tumors were imaged by MRI after 5 weeks, and drug intervention was initiated for tumors ≥2 mm3. A total of 90 animals were treated in long-term experiments with BLZ945 alone, which represented 5 independent cohorts. These data are compiled and presented in
p53 KD Mouse Model.
Injections were performed as described for the PDG mouse model above, except Ntv-a mice were used (i.e. WT Ink4a/Arf). Mice were intracranially injected with a 1:1 ratio of DF-1:RCAS-hPDGF-B-HA cells and DF-1:RCAS-shp53 cells (total of 300,000 cells/2 □l) between 5-6 weeks of age. Injection into the SVZ in the p53 knockdown (KD) model induces high-grade tumors with low latency (6-7 weeks), 100% penetrance, and histological features of human GBM (13, 54, 55). Tumors were detected by Mill after 6-7 weeks, at which point drug intervention with BLZ945 was initiated (see
Pten KO Mouse Model.
Injections were performed as described for the PDG mouse model above, except Ntv-a; Ptenflox mice were used (i.e. WT Ink4a/Arf). Mice were intracranially injected with a 1:1 ratio of DF-1:RCAS-hPDGF-B-HA cells and DF-1:RCAS-Cre cells (total of 300,000 cells/2 μl) between 5-6 weeks of age. Injection into the SVZ in the Pten knockout (KO) model induces tumors with moderate latency (8-12 weeks) and penetrance (˜20-30%). Tumors that form harbor key characteristics of human GBM including highly infiltrative histology (13, 54, 56, 57). Tumors were detected by Mill after 8-12 weeks, at which point drug intervention with BLZ945 was initiated (see
Derivation of Mouse Primary Glioma Cell Lines from PDG Tumors.
To derive rebound or dormant cell lines from BLZ945-treated PDG tumors, MRI was used to confirm whether a particular tumor was in rebound or dormancy phase. Macro-dissected rebound or dormant lesions from the BLZ945-treated PDG mouse model were manually dissociated and filtered through a 40 μm mesh filter. The cell suspension was washed 2× with PBS, and cultured in Mouse Neural Stem Cell (mNSC) Basal Media (Stem Cell Technologies) containing mNSC proliferation supplement, 1 mg/ml Heparin (Stem Cell Technologies), 10 ng/ml recombinant epidermal growth factor (rEGF; Invitrogen), and 20 ng/ml recombinant basic-fibroblast growth factor (rbFGF; Sigma). To generate cell lines in monolayer, tumorsphere cultures were expanded and dissociated, and transferred to culture with DMEM+10% FBS (59). In total, 5 rebound cell lines were derived (89AReb, 89BReb, 74Reb, 48Reb, 52Reb), and 1 cell line was derived from the 28d dormant timepoint. The 28d dormant cells (
Human Cell Lines.
Human umbilical vein endothelial cells (HUVEC) were obtained from ATCC. Human brain microvascular endothelial cells (HBMEC) and human astrocytes were purchased from Sciencell. Astrocytes were cultured on poly-L-lysine-coated plates, and both HUVECs and HBMECs were cultured on gelatin-coated plates with endothelial cell media (ECM, Sciencell)+10% FBS+an endothelial cell growth factor supplement. The U251 (commercially available) and TS573 (patient-derived) cell lines were selected based on previously published work, which showed efficacy in response to BLZ945 in orthotopic xenograft trials (8). The patient-derived TS573 glioma tumorsphere line was derived from a consenting patient under Institutional Review Board (IRB)-approved protocols for the banking of excess tumor tissue during routine surgical resection, as previously described (8, 60). Tumorspheres were maintained in Human Neural Stem Cell (hNSC) Basal Media (Stem Cell Technologies) containing hNSC proliferation supplement, 1 mg/ml Heparin (Stem Cell Technologies), 10 ng/ml rEGF (Invitrogen), and 20 ng/ml rbFGF (Sigma). Tumorspheres were passaged by dissociation with Accutase cell detachment solution (Millipore). Characterization and molecular subtyping by Sequenom, Nanostring and aCGH were performed as previously described (8). Briefly, aCGH on primary spheroids showed a high level amplification of PDGFRA and CDK6 loci, and a regional chromosome 5 loss. Nanostring analysis confirmed PDGFRA overexpression, and sequenom analyses were negative for IDH1/2 mutations.
Isolation of Bone Marrow-Derived Macrophages (BMDMs).
To generate macrophages from bone marrow, femurs and tibiae from Stat6−/−, Il4raflox; LysM-cre, or WT BL6 mice were flushed and cells harvested under sterile conditions. The isolate was filtered through a 40 μm mesh filter and cultured in 30 ml Teflon bags (PermaLife PL-30) for 5-7 days in DMEM+10% FBS+10 ng/ml recombinant mouse CSF-1 (R&D Systems). Media were changed every other day.
TGL Infections.
Cell lines were labeled with a triple-imaging vector (TK-GFP-Luc; TGL) (61) for use in orthotopic in vivo experiments. The TGL vector was developed to enable non-invasive in vivo imaging of tumor growth over time. A standard protocol for retroviral infection was used. Briefly, GP2-293T cells were transfected with the TGL construct and pCL-Ampho at a 1:1 ratio, using Fugene (Promega) and OptiMEM (Gibco). 12 h later, media was replaced with complete antibiotic-free DMEM, and collected for 3 consecutive days for infection of target cells.
Orthotopic Transplantation Experiments.
TGL-labeled cells were resuspended in antibiotic-free serum-free DMEM for all orthotopic injections. Mice were fully anesthetized with ketamine/xylazine prior to surgery. Pain management included a 501 subcutaneous injection of bupivacaine (0.25%) at the surgical site prior to surgery, and an intraperitoneal injection of buprenorphine immediately following surgery. Mice were intracranially injected with glioma cells between 5-6 weeks of age using a fixed stereotactic apparatus (Stoelting). The number of cells injected was as follows: mouse glioma lines 2.5-5×104 cells/2 μl, human U251 cells 2.5×105 cells/2 μl, and patient-derived TS573 cells 5×104 cells/2 Injections were made to the right frontal cortex, approximately 1.5 mm lateral and 1 mm caudal from bregma, and at a depth of 2 mm. Hydrogen peroxide was used to clean the hole made by the surgical drill, and bone wax was used to close the hole. The incision was sealed using Vetbond tissue adhesive (3M). One week following injections, mice were randomly assigned to Vehicle or BLZ945 treatment groups, and dosed 1× daily by oral gavage. Bioluminescence imaging (BLI; Xenogen IVIS-200 Optical In Vivo Imaging System) was performed every 3-5 days over the course of the experiment to monitor tumor progression and response to therapy. Once BLZ945-treated tumors reached 3× the volume of their lowest BLI measurement, tumors were considered ‘resistant’, and mice were randomly assigned to combination treatment with BLZ945+OSI906, or BLZ945+vehicle. For doxycycline (dox)-inducible IGF1R shRNA experiments with U251 cells, mice were additionally assigned to either +/− dox groups (doxycycline hyclate diet formulated at 2,500 mg/kg, Envigo).
Animal Euthanasia and Tissue Harvest.
Mice were euthanized at the defined 26 week endpoint, or when symptomatic (poor grooming, lethargy, weight loss, hunching, macrocephaly/hydrocephalus, seizures). 26 weeks was selected because mice in the Ink4a/Arf−/− background develop spontaneous lymphomas and sarcomas beginning at ˜30 weeks of age (62). Euthanasia was performed by either carbon dioxide asphyxiation or anesthesia (avertin; 2,2,2-tribromoethanol; Sigma) followed by cervical dislocation. For snap freezing of whole tumor samples, mice were euthanized 1 h following the last treatment dose, and tissues were collected, frozen immediately in liquid nitrogen, and stored at −80° C. for subsequent applications (e.g. RNA isolation, protein extraction). For isolation of whole tissues for histology, mice were fully anesthetized with avertin, transcardially perfused with 10 ml of PBS, followed by 10 ml of paraformaldehyde (PFA; 4% in PBS). Tissues were incubated in PFA overnight at 4° C., rinsed in PBS, and then transferred to sucrose (30%) for 2-3 days at 4° C. For hypoxia analysis, mice were injected intraperitoneally with 60 mg/kg of pimonidazole (hypoxyprobe-1; HPI) ˜20 min prior to sacrifice and tissue collection. All tissues were embedded and frozen in Optimal Cutting Temperature (OCT) compound (Tissue-Tek®), and 10 μm cryostat tissue sections were used for all subsequent staining and analyses.
Immunofluorescence (IF).
For IF staining, 10 μm frozen sections were thawed and dried at room temperature, and rinsed in PBS. Tissue sections were blocked in 0.5% Blocking Reagent (PerkinElmer; 1 h at room temperature or overnight at 4° C.), followed by incubation in primary antibody (2 h at room temperature or overnight at 4° C.). Sections were then washed in PBS and incubated with the appropriate fluorophore-conjugated secondary antibody (Molecular Probes) at a dilution of 1:500 in 0.5% PNB (1 h at room temperature). DAPI was used as a counterstain prior to mounting with fluorescent mounting media (Dako).
Immunohistochemistry (IHC) and Von Kossa Staining.
For manual IHC, tissue sections were first subjected to citrate buffer based antigen retrieval by submerging in antigen unmasking solution (0.94% v/v in distilled water; Vector Laboratories) and microwaving for 10 min, followed by cooling to room temperature for 30 min. Endogenous peroxidases were blocked for 10 min with Dual Endogenous Enzyme Block (Dako). Slides were incubated with serum-free protein block (Dako) for 1 h at room temperature, and then incubated with primary antibody in a humidity chamber overnight at 4° C. The following day, slides were washed and incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch) for 1 h at room temperature, and positive staining was detected using diaminobenzidine (DAB) substrate-chromogen. Haematoxylin was used as a counterstain and slides were mounted with VECTASHIELD® mounting media (Vector). As an alternative to manual staining, a Ventana autostainer was used for staining of mouse GFAP, human phospho (p)-AKT, and human MRC1, which included automated deparaffinization, citrate buffer-based antigen retrieval, non-specific protein and endogenous peroxidase block, antibody incubation, and DAB detection. For visualization of calcium deposition in tissue sections, a Von Kossa staining kit was used as per manufacturer's instructions (Abcam). Briefly, tissues are treated with a silver nitrate solution, which is deposited by replacing calcium reduced by UV light. Nuclear fast red was used as a counterstain (Vector).
Histology and Grading.
For analysis of tissue histology and grading of tumor malignancy, haematoxylin and eosin (H&E) staining was performed using a Tissue-Tek® automated slide stainer, and slides were mounted with VECTASHIELD® mounting media (Vector). Tissues were blindly graded by a neuropathologist, according to standard WHO criteria (63). For CNS tumors, this grading system is based on a malignancy scale, where tumors that are minimally proliferative and infiltrative are considered grade I, while the most histologically aggressive, infiltrative, and incurable tumors (glioblastomas) are grade IV (63). GFAP quantification was performed by the Allred scoring method, which is the sum of a proportion score (0-5) and an intensity score (0-3) for a given marker (64).
Image Analysis.
Stained tissue sections were visualized under a Carl Zeiss Axioimager Z1 microscope equipped with an ApoTome.2, or a Carl Zeiss Axioimager M1 epifluorescence and brightfield microscope. Staining analyses were performed using a TissueGnostics slide scanning platform, and TissueQuest analysis software. This automated analysis platform was used to quantify number of positive counts, area of positive staining, and/or microvascular density within stained tissue sections in an unbiased manner (8).
Patient GBM Tissue Samples.
Patient GBM tissue samples used for staining and analysis (
Protein Isolation and Immunoblotting.
To evaluate phospho-protein status of IGF-1R and AKT in culture, cells were seeded at 70-80% confluence, serum-starved for ˜12 h, and then treated with OSI906 for 30 min. To evaluate phosphorylation of IGF-1R or AKT in snap-frozen tumor samples, tissues were dissociated with a glass homogenizer on ice in the presence of lysis buffer. All protein lysates were prepared in RIPA lysis buffer supplemented with Halt™ protease inhibitor (1:100; Thermo Scientific) and Halt™ phosphatase inhibitor (1:100; Thermo Scientific), and protein was quantified using a BCA protein assay kit (Pierce). Equal amounts of protein (20 μg/lane for cell lines, and 100 μg/lane for tissue samples) were loaded onto SDS-PAGE precast gels (Invitrogen) and transferred to PVDF membranes for immunoblotting. Membranes were blocked in 5% milk, incubated with primary antibodies (Table S4) for 1 h at room temperature or overnight at 4° C., washed with 0.1% TBS-T, and incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch) for 1 h at room temperature. SuperSignal West Femto or Pico chemiluminescent substrate and CL-XPosure Film (Pierce) were used for signal detection.
RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR.
RNA was isolated with Trizol, DNase treated, and 1 μg of RNA was used for cDNA synthesis using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Mouse taqman probes (Applied Biosystems) were used for quantifying expression of Igf1 (Mm00439560_m1), Chil3 (Mm00657889_mH), Ccl17 (Mm01244826_g1), Retnla (Mm00445109_m1), Il4 (Mm00445260_m1), Arg1 (Mm00475988_m1), Cd36 (Mm00432403_m1), Mrc1 (Mm01329362_m1), Ubc (Mm02525934_g1; housekeeping), and Hprt (Mm01545339_m1; housekeeping). Human taqman probes (Applied Biosystems) were used for quantifying expression of IL4 (Hs00174122_m1), IL13 (Hs00174379_m1), IGF1 (Hs01547656_m1) and HPRT1 (Hs02800695_m1; housekeeping).
MTT Assays.
Cell growth rate was determined using an MTT cell proliferation kit (Roche). Briefly, cells were plated in triplicate in 96-well plates. 1×103 cells/well were plated for mouse glioma cell lines, and 5×103 cells/well were plated for BMDMs. For BLZ945 time course experiments, cells were grown in the presence of 6,700 nM of BLZ945 versus an equal percent DMSO, media was changed every 48 h, and viability measurements were taken every 24 h. For dose response experiments, cells were grown in the presence of an IGF-1R inhibitor (AEW541, ADW742, BMS754807, or OSI906) versus an equal percent DMSO at the doses indicated (see “Biologicals and pharmaceuticals” section above), and viability measurements were taken after 24 h. Reduction of the MTT substrate was detected by colorimetric analysis using a plate reader as per the manufacturer's protocol. 10 μl of MTT labeling reagent was added to each well and then incubated for 4 h at 37° C., followed by the addition of 100 μl MTT solubilization reagent overnight. The mixture was gently resuspended and absorbance was measured at 595 nm and 750 nm on a spectraMax 340pc plate reader (Molecular Devices).
Ex Vivo GMEC Assays.
Glioma microenvironment cultures (GMECs) are early-passage heterotypic cell cultures harvested directly from PDG mouse primary tumors. Flow cytometry characterization of these cultures revealed that passage 1 GMECs contain a high abundance of tumor cells, astrocytes, and macrophages, as well as smaller proportions of myeloid progenitors, T cells and B cells (
Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS) on Primary Mouse Tissues.
Mice were fully anesthetized with avertin and transcardially perfused with 20 ml of PBS. The brain was then isolated and the tumor was macrodissected from the surrounding normal tissue. Tissues were mechanically dissociated and filtered into a single cell suspension. For flow cytometry, cells were counted and incubated with Fc block for 1 h (BD Biosciences; 1:100/106 cells), followed by a 30 min incubation with LIVE/DEAD® fixable dead cell kit (Invitrogen), and then a 1 h incubation with conjugated antibodies for extracellular markers. For FACS, cells were counted and incubated with Fc block for 1 h, followed by a 1 h incubation with conjugated antibodies, and then stained with DAPI for dead cell exclusion. OneComp eBeads (eBioscience), ArC™ Amine Reactive Compensation Beads (Invitrogen), and/or cell suspensions from spleen were used for compensation controls. A BD LSRFortessa™ was used for flow cytometry, and a BD FACSAria III™ was used for cell sorting.
Distinguishing Between Putative BMDMs and Microglia by Flow Cytometry.
Flow cytometry was used to evaluate the proportions of peripherally derived BMDMs versus resident microglia in PDG tumors across different treatment groups, according to published methods (22-24). Briefly, after gating on live cells, we used cell surface expression of CD45 and CD11b to distinguish between the two populations, where CD45lo CD11b+ defined putative microglia, while CD45hi CD11b+ defined putative BMDMs. In using this method, we acknowledge its limitations, and recognize that these two populations cannot be definitively distinguished without lineage tracing experiments that specifically label yolk sac-derived microglia or peripherally recruited BMDMs.
FACS Purification of Human Peripheral Immune Cell Types.
Human buffy coats from three consenting healthy donors were obtained from the New York Blood Center. For isolation of neutrophils and eosinophils, buffy coats were directly RBC lysed (BD PharmLyse) for 15 minutes at room temperature. All other cell types were isolated from the top layer of a Ficoll gradient separation (HistoPaque, Sigma). Cells were pelleted for 10 minutes at 300×G and washed twice with FACS buffer (PBS+2% fetal bovine serum) and Fc blocked (Biolegend TruStain FcX). Cells were incubated with the appropriate antibodies for 15 minutes (Table S4). Cells were FACS purified on an Aria III (BD). For human macrophage differentiation, PBMCs were isolated from buffy coats following a Ficoll gradient. Monocytes were further purified from the interphase of a 70%/30% Percoll gradient. Monocytes were then washed twice with PBS, and cultured in Teflon bags (Origin) for 7 days in DMEM+2% human serum+recombinant human CSF-1 (10 ng/ml; R&D Systems). CSF-1 and media were replaced every 48 h.
Array Comparative Genomic Hybridization (aCGH).
All sequencing and quality control was performed at the Integrated Genomics Operation, MSKCC. DNA was isolated from passage 1 PDG neurospheres from rebound tumors or corresponding liver tissue using TRIzol as per manufacturer instructions (Invitrogen). 3 ug of DNA was used with an Agilent standard cy5/cy3 labeling protocol. Briefly, Agilent Mouse CGH 180 k arrays were hybridized at 65° C. and 20 rpm for 40 h. Slides were then scanned using the Agilent scanner according to the manufacturer's instructions. The raw data were extracted with Feature Extraction using Agilent default analysis settings. Subsequent analyses were performed in R v3.1.0 using the “DNAcopy” package (65).
RNA-Sequencing.
Three RNA-seq experiments were performed in total: (i) FACS-purified tumor cells (CD45-PDGFRα+) and TAMs (CD45+CD11b+Gr1−) from Veh, EP and Reb tumors, (ii) FACS-purified astrocytes (CD45-GLAST+), B cells (CD45+CD19+), Tc cells (CD45+CD3+CD8+) and bulk T cells (CD45+CD3+CD8−) from Reb tumors, (iii) FACS-purified TAMs (CD45+CD11b+Gr1−) from 28d and Reb tumors. RNA-sequencing and quality control was performed at the Integrated Genomics Operation, MSKCC, or GENEWIZ, N.J. In all cases, RNA was isolated using TRIzol as per manufacturer instructions (Invitrogen), and RNA integrity was assessed by an Agilent Bioanalyzer 2100. RNA-sequencing libraries were prepared using the SMART-Seq library preparation kit and 2×50 or 2×100 base pair sequencing was performed on an Illumina HISeq 2000. Sequencing quality was assessed with FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the mouse genome (mm10) using STAR 2.3.0e (66) with the default parameters, a minimum intron length of 70 base pairs and a maximum of 100,000 base pairs. BAM files were generated and sorted, duplicate reads were then removed using SAMTOOLS (67). Read counts were tabulated with HT-Seq using “union” mode and the iGenomes GFF file as a reference (Illumina) (68).
Gene Expression Analyses.
Raw count data from HT-Seq was imported into R (v3.1.0) and normalized using limma voom (69). Principal component analysis was completed using the princomp function on mean centered data. A log 2 fold change cutoff of 1 and a false discovery rate of 10% were applied for all differential gene expression analyses (Table S1). Significantly upregulated genes from these lists were used in gene ontology analyses using DAVID (70). Gene set variation analysis (GSVA) was performed on RNA-seq data from FACS-purified PDGFRα+ tumor cells, using the GSVA package (14) with gene sets from the C2 group from MSigDB (71). A log 2 fold change cutoff of 1 and a false discovery rate cutoff of 10% were used to determine differentially enriched gene sets (
Transcription Factor Activity Analysis.
Transcription factor (TF) activity analysis was performed as an adaptation of previously published methods: ISMARA (72) and Regulatorinference (73). Briefly, transcription start sites and Motevo predictions (74) of binding sites were downloaded from the Swiss Regulon (http://swissregulon.unibas.ch/fcgi/sr/downloads#). These were used to determine the number of predicted binding sites for 185 transcription factor families across all mouse promoters. Promoters were designated as 2 kilobases upstream and downstream of transcription start sites. This tabulated matrix was then used in a ridge regression to model log 2 gene expression values generated by voom. Ridge regression was performed with the glmnet function in R (75). The regularization parameter, lambda, was identified for each sample through 10-fold cross validation. The coefficients for each TF family were z-scored and used as relative TF activity scores in subsequent analyses. Differentially enriched TFs were identified by using the z-scored values in limma with a log 2 fold change cutoff of 1 and a false discovery rate of 10% (Table S3).
External Data Set Analysis.
RNA-seq expression data from the TCGA glioblastoma patient data set was downloaded using TCGA-assembler (76). For survival analyses, these data were filtered for patients with updated clinical information from the Broad Firehose. Correlations between IGF1 and macrophage markers (CD163, MRC1, CSF1R, CD68, AIF1) or astrocyte markers (GFAP and ALDH1L1) were assessed using a Spearman correlation coefficient. Normalized gene expression data for human bulk tumor versus tumor-associated macrophage fraction was downloaded from the GEO under accession number GSE16119 (44). Subtype calls for patients were obtained from GlioVis (http://gliovis.bioinfo.cnio.es). PI3K signature scores were tabulated with a single sample gene set enrichment, as used for macrophage activity analysis. The gene set was from the Hallmark collection from MsigDB, systematic name M5923 (43).
Data Availability.
All RNA-seq data has been deposited to the GEO under the accession number GSE69104, and aCGH data under GSE80399. All code used in this project can be found at https://bitbucket.org/bowmanr/joycelab-brain-tme.
Data Presentation and Statistical Analysis.
GraphPad Prism 6.0 or R Studio was used for all data analysis. Parametric data are presented as mean±standard error (s.e.m.) and were analyzed by an unpaired two-tailed Student's t-test. For multiple comparisons, a one-way ANOVA with Tukey's or Dennett's correction was used as noted in the figure legend. Non-parametric data were analyzed by a Mann-Whitney test on ranks. For survival curves, P-values were obtained using the Log Rank (Mantel-Cox) test. Fisher's exact test was used for histological tumor grading. A pairwise Spearman correlation test was used for correlational analyses. P<0.05 was considered as statistically significant in all cases. Principal component analyses, correlation plots, Volcano plots, heatmaps, and network plots were plotted in R Studio using base graphics (http://www.R-project.org/), rg1 (http://CRAN.R-project.org/package=rg1), gplots (http://CRAN.R-project.org/package=gplots), ggplot2 (77) and qgraph packages (78).
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Claims
1. A method of treating CSF-1R inhibitor resistant glioma, the method comprising administering to a mammalian subject with a CSF-1R inhibitor resistant glioma an effective amount of: (a) a CSF-1R inhibitor, and (b) an IGF-1R inhibitor or a PI3K inhibitor, thereby treating the CSF-1R inhibitor resistant glioma.
2. The method of claim 1, comprising administering to the subject an effective amount of: (a) a CSF-1R inhibitor, (b) an IGF-1R inhibitor, and (c) a PI3K inhibitor.
3. The method of claim 1, wherein the CSF-1R inhibitor is selected from the group consisting of BLZ945, GW2580, ABT-869 (linifanib), OSI-930, CEP-32496, AC708, PLX3397, AZD6495, CYC10268, IMC-CS4, RG7115, pyridyl bisamides, thiazolyl bisamides, 6-O-substituted benzoxazoles, and 6-O-substituted benzothiazoles.
4. The method of claim 1, wherein the IGF-1R inhibitor is selected from the group consisting of OSI906 (linsitinib), NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, picropodophyllin (PPP), GSK1838705A, AG-1024, PQ401, and BMS-754807.
5. The method of claim 1, wherein the PI3K inhibitor is selected from the group consisting of BKM120, idelaisib, SAR245409, SAR245408, BYL-719, GDC-0980, GDC-0941, wortmannin, Ly294002, demethoxyviridin, perifosine, delalisib, PX-866, IPI-145, BAY 80-6946, BEZ235, RP6530, TGR 1202, RP5264, SF1126, INK1117, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477, CUDC-907, and AEZS-136.
6. The method of claim 1, wherein if either the CSF-1R inhibitor, the IGF-1R inhibitor, or the PI3K inhibitor is able to cross the blood brain barrier it is administered to the subject systemically, and if either the CSF-1R inhibitor, the IGF-1R inhibitor, or the PI3K inhibitor is not able to cross the blood brain barrier it is administered to the subject intracranially.
7. The method of claim 1, wherein administration of the IGF-1R inhibitor and/or the PI3K inhibitor is commenced either (a) at approximately the same time as administration of the CSF-1R inhibitor is commenced, (b) after administration of the CSF-1R inhibitor is commenced, or (c) after the subject has developed resistance to CSF-1R inhibitor treatment.
8. The method of claim 1, wherein the subject was previously treated with a CSF-1R inhibitor.
9. The method of claim 1, wherein the glioma is a glioblastoma.
10. The method of claim 9, wherein the glioblastoma is selected from the group consisting of proneural glioblastoma, Glioblastoma Multiforme (GBM), astrocytoma and oligodendroglioma.
11. The method of claim 1, wherein the mammalian subject is selected from the group consisting of a rodent, a mouse, a non-human primate, and a human.
12. The method of claim 1, wherein the mammalian subject is a human.
13. The method of claim 1, wherein the subject is also treated by surgery, radiation therapy, chemotherapy, or anti-angiogenic therapy.
14. The method of claim 1, wherein the CSF-1R inhibitor is BLZ945.
15. The method of claim 1, wherein the IGF-1R inhibitor is OSI906.
16. The method of claim 1, wherein the PI3K inhibitor is BKM120.
17. The method of claim 1, comprising administering to the subject an effective amount of: (a) the CSF-1R inhibitor BLZ945, and (b) the IGF-1R inhibitor OSI906 or the PI3K inhibitor BKM120, thereby treating the CSF-1R inhibitor resistant glioma.
18. The method of claim 1, comprising administering to the subject an effective amount of: (a) the CSF-1R inhibitor BLZ945, and (b) the IGF-1R inhibitor OSI906, thereby treating the CSF-1R inhibitor resistant glioma.
19. The method of claim 1, comprising administering to the subject an effective amount of: (a) the CSF-1R inhibitor BLZ945, and (b) the PI3K inhibitor BKM120, thereby treating the CSF-1R inhibitor resistant glioma.
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Type: Grant
Filed: May 9, 2016
Date of Patent: Jul 28, 2020
Patent Publication Number: 20180125852
Inventors: Daniela Francis Quail (Montreal), Johanna Alexandra Joyce (Lausanne), Robert Lyle Bowman (New York, NY)
Primary Examiner: Theodore R. West
Application Number: 15/572,456
International Classification: A61K 31/428 (20060101); A61K 31/4985 (20060101); A61K 31/5377 (20060101); A61P 35/00 (20060101); A61K 45/06 (20060101); A61K 31/4439 (20060101); A61P 25/00 (20060101); A61K 31/416 (20060101); A61K 31/4184 (20060101); A61K 31/423 (20060101); A61K 31/426 (20060101); A61K 31/437 (20060101); A61K 31/4406 (20060101); A61K 31/47 (20060101); A61K 31/496 (20060101); A61K 31/517 (20060101); A61K 31/519 (20060101);
